an epigenetic regulator emerges as microtubule minus-end ... · received 16 jan 2015 | accepted 23...

ARTICLE

Received 16 Jan 2015 | Accepted 23 Jun 2015 | Published 5 Aug 2015

An epigenetic regulator emerges as microtubuleminus-end binding and stabilizing factor in mitosisSylvain Meunier1,2,*, Maria Shvedunova3,4,*, Nhuong Van Nguyen3,4, Leonor Avila1,2, Isabelle Vernos1,2,5

& Asifa Akhtar3

The evolutionary conserved NSL complex is a prominent epigenetic regulator controlling

expression of thousands of genes. Here we uncover a novel function of the NSL complex

members in mitosis. As the cell enters mitosis, KANSL1 and KANSL3 undergo a marked

relocalisation from the chromatin to the mitotic spindle. By stabilizing microtubule minus

ends in a RanGTP-dependent manner, they are essential for spindle assembly and chromo-

some segregation. Moreover, we identify KANSL3 as a microtubule minus-end-binding pro-

tein, revealing a new class of mitosis-specific microtubule minus-end regulators. By adopting

distinct functions in interphase and mitosis, KANSL proteins provide a link to coordinate the

tasks of faithful expression and inheritance of the genome during different phases of the cell

cycle.

DOI: 10.1038/ncomms8889 OPEN

1 Cell and Developmental Biology Programme, Centre for Genomic Regulation (CRG), Dr Aiguader 88, 08003 Barcelona, Spain. 2 Universitat Pompeu Fabra(UPF), Dr Aiguader 88, 08003 Barcelona, Spain. 3 Max Planck Institute of Immunobiology and Epigenetics, Department of Chromatin Regulation,79108 Freiburg im Breisgau, Germany. 4 Faculty of Biology, University of Freiburg, 79104 Freiburg im Breisgau, Germany. 5 Institucio Catalana de Recerca iEstudis Avancats (ICREA), Passeig Lluıs Companys 23, 08010 Barcelona, Spain. * These authors contributed equally to this work. Correspondence andrequests for materials should be addressed to I.V. (email: [email protected]) or to A.A. (email: [email protected]).

NATURE COMMUNICATIONS | 6:7889 | DOI: 10.1038/ncomms8889 | www.nature.com/naturecommunications 1

& 2015 Macmillan Publishers Limited. All rights reserved.

mailto:[email protected]

mailto:[email protected]

http://www.nature.com/naturecommunications

Chromatin modifiers are responsible for the establishmentof ‘chromatin states’ that determine the accessibilityand activity of nuclear DNA. Different chromatin states

are associated with altered organization and compaction ofchromatin, and determine a given locus’ transcriptional output.

The heptameric KAT8-associated nonspecific lethal (KANSL(mammals) or NSL (Drosophila); for simplification we will use‘KANSL’ throughout) complex is an important chromatinmodifier in higher animals. Previous studies have determinedthat the KANSL proteins interact robustly both in vitro andin vivo and show high evolutionary conservation1–3. InDrosophila cells, the proteins are prominent regulators ofhousekeeping genes, with 89.4% of constitutively expressedgenes bound by at least one KANSL protein4–6. In mammalianembryonic stem cells, these proteins additionally regulateenhancers and appear to be important for proliferation7,8.

KANSL proteins are essential in all the species in which theyhave been studied. Drosophila KANSL null mutants die latest inlarval stages, and likely only survive thus far due to maternalcontribution6. Misregulation of these proteins is additionallyassociated with diverse disease states in humans. A heterozygousmutation of the KANSL1 locus is sufficient to manifestin the Koolen-de Vries/KANSL1-related intellectual disabilitysyndrome9–11 and mosaic single-nucleotide variations inKANSL2 were found to be frequently associated with severeintellectual disability in patients12. Thus far, the only knownfunctions of the NSL complex have been described ininterphase5,7.

During mitosis, a global rearrangement of chromatin structuretakes place, leading to a totally unique chromatin state, the highlycondensed ‘mitotic chromatin’. Although some chromatinmodifiers remain associated with chromatin in mitosis, the vastmajority of these factors are evicted, freeing them to performfunctions in other cellular compartments13,14. In parallel, somenuclear proteins are known to play an essential role in theassembly of the mitotic spindle, by promoting microtubulenucleation and stabilization at the vicinity of the chromosomes15.This process is dependent on the small GTPase Ran, whose activeGTP-bound form is concentrated around the mitoticchromatin16. A number of RanGTP-regulated spindle assemblyfactors have been identified so far17,18, including Imitation Switch(ISWI), which functions as a nucleosome remodeler ininterphase19. It remains unclear how many other epigeneticcomplexes may have functions in mitosis not related tochromatin states or gene expression.

In this study, we describe the essential and novel contributionof KANSL1 and KANSL3 to spindle assembly. We found thatKANSL1 and KANSL3 are novel microtubule-associated proteinsthat localize to the spindle poles during mitosis. Using Xenopusegg extracts to study their transcription-independent functions,we show that they interact with TPX2 in a RanGTP-dependentmanner, promoting microtubule assembly in vitro. Moreover, weshow that KANSL3 is an autonomous microtubule minus-end-binding protein. Our results suggest that KANSL3 targets otherNSL complex members to K-fibre minus ends regulating theirdynamics and performing an essential role in spindle assembly.

ResultsKANSL1 and KANSL3 are required for cell division. Toinvestigate whether the NSL complex could have additional rolesin mitosis, we decided to focus on two of its core components,KANSL1 and KANSL3. To ascertain whether expression ofNSL complex proteins is cell cycle regulated, we synchronizedcells in discrete phases of the cell cycle with specific inhibitorsand probed protein levels by western blot. We observed that

KANSL1 and KANSL3 protein levels remained constantthrough all cell cycle stages (Fig. 1a; Supplementary Fig. 1a).Co-immunoprecipitation (co-IP) experiments in nocodazole-synchronized HeLa cells further showed that KANSL proteinswere able to interact equally efficiently in mitosis as they do ininterphase cells (Supplementary Fig. 1b).

Next, we investigated whether loss of NSL complex proteinshad a consequence for mitotic progression. For this purpose, weused a cell line expressing mCherry-marked histone 2B and greenfluorescent protein (GFP)-tagged tubulin20. We transfected cellswith short interfering RNA (siRNAs; Supplementary Fig. 1c) andrecorded stacks of images every 3 min over a 24-h period.Knockdown of KANSL1 or KANSL3 resulted in marked mitoticdefects, the most prevalent of which was a prolonged arrest ofcells in a prometaphase-like state (Fig. 1b; Supplementary Fig. 1d;Supplementary Movies 1–3). Following KANSL1 knockdown,61% of cells entering mitosis were not able to complete it duringthe 24-h recording period (Fig. 1c). Cells silenced for KANSL3had a less marked phenotype, possibly because it does notdestabilize other members of the NSL complex such asKANSL1 silencing does (Fig. 1c; Supplementary Fig. 1c).A large proportion of KANSL1 and KANSL3 siRNA-treatedcells exhibited misaligned chromosomes and various spindleorganization defects (see Supplementary Movies andSupplementary Fig. 1e). The similarities of the phenotypesobserved on knockdown of the two NSL complex memberssuggested a common function for these proteins during mitosis(Fig. 1b,c; Supplementary Fig. 1d,e; Supplementary Movies 4–6).

Having observed mitotic defects on their knockdown, we thenused antibodies against KANSL1 and KANSL3 to investigate theirsubcellular localizations. In interphase, both the proteins wereconcentrated in the nucleus as expected (Fig. 1d, top panel).Interestingly, KANSL1 and KANSL3 exhibited a markedrelocalisation during mitosis (Fig. 1d). In prometaphase andmetaphase, KANSL1 was concentrated at spindle poles and in thepericentriolar region. The protein appeared then to remainassociated with spindle poles throughout anaphase (Fig. 1d).Similarly to KANSL1, KANSL3 was strongly enriched at thespindle poles throughout mitosis (Fig. 1d). Staining specificitywas verified by knockdowns (Supplementary Fig. 1f).

KANSL1 and KANSL3 promote microtubule assembly in vitro.The experiments above strongly suggested a role for KANSL1 andKANSL3 in mitosis. However, since the KANSL proteins regulateexpression of housekeeping genes4,5,7, to separate this interphasefunction from a direct role in mitosis, we decided to takeadvantage of the Xenopus laevis egg extract system, in whichtranscription is totally inhibited21. Moreover, most of the corecomponents of the NSL complex have been recently identifiedin a Xenopus laevis egg proteomic analysis22. We used threemembers of the Drosophila NSL complex that could be expressedand purified as full-length soluble proteins: 3FLAG/HA-taggedDrosophila KANSL1, KANSL3 and males absent on the first(MOF) (Fig. 2a). The proteins were individually incubated in eggextract either with or without exogenous RanGTP, and recoveredby immunoprecipitation on magnetic beads. The beads were thenwashed and used for western blot analysis or placed in puretubulin to test their microtubule stabilization or nucleationactivities (Fig. 2b).

Interestingly, KANSL1 and KANSL3 beads, but not MOFbeads, pulled down two important spindle assembly factors,TPX2 and MCAK, in a RanGTP-dependent manner (Fig. 2c).Moreover, the KANSL1 and KANSL3 beads retrieved from anextract containing RanGTP, but not from a control extract,promoted microtubule assembly in pure tubulin (Fig. 2d).

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8889

2 NATURE COMMUNICATIONS | 6:7889 | DOI: 10.1038/ncomms8889 | www.nature.com/naturecommunications



In contrast, MOF-coated beads were unable to inducemicrotubule assembly in the same experimental conditions(Supplementary Fig. 2a). Since TPX2, a well-characterized mitoticprotein, is known to be required for RanGTP-dependentmicrotubule nucleation23, we tested whether it was responsiblefor the microtubule assembly observed. We found that KANSL1or KANSL3 beads retrieved from TPX2-depleted extractscontaining RanGTP did not promote microtubule assemblyin vitro (Fig. 2d). TPX2 is therefore essential for microtubulenucleation from KANSL1 and KANSL3 beads. However,incubation of KANSL1 or KANSL3 beads with an excess ofimportin-b abolished their microtubule assembly activity in thisassay (Fig. 2d). Since importin-b does not bind directly to TPX2and therefore does not inhibit its microtubule nucleation-promoting activity24, this indicates that other RanGTP-regulated protein(s) present on the beads additionallyparticipate in the assembly of microtubules from KANSL1 andKANSL3 beads in vitro. We conclude that KANSL1 and KANSL3,in complex with spindle assembly factors, promote microtubuleassembly in a RanGTP-dependent manner. This strongly suggeststhat both KANSL1 and KANSL3 have additional functions in

mitosis related to microtubules and spindle assembly thatare independent of their interphase role in transcriptionregulation.

KANSL1 and KANSL3 regulate K-fibre minus-end dynamics.The RanGTP-dependent microtubule assembly activity of theKANSL proteins revealed by the egg extract experiments wasreminiscent of the results described for an interphase KANSL-interacting protein, MCRS1 (ref. 25). However, MCRS1 alsoassociates with other chromatin complexes in interphase1 and it isnot known whether any of these interactions are maintained inmitosis. We therefore investigated this issue using a stable isotopelabelling of amino acids in cell culture (SILAC)-based quantitativeproteomic approach to identify MCRS1 mitotic partners. Fiveproteins from the NSL complex scored as specific partners ofMCRS1 (Supplementary Fig. 2b). We confirmed these data by co-IP experiments from synchronized cells (Supplementary Fig. 2c).Consistently with our results in egg extract (above and ref. 25),we found that KANSL1, KANSL3 and MCRS1 also interact withTPX2 during mitosis in mammalian cells (Supplementary Fig. 2c).

siS

CR

AM

BLE

Dsi

KA

NS

L3

0 15 30 45 60

4230 30 60

0 30 60 420 780

siK

AN

SL1

Prometa-phase

Metaphase

Anaphase

b

d

H2BTubulin

KANSL1 Tubulin Merge

TubulinKANSL1

DNA DNA

KANSL3 Tubulin Merge

TubulinKANSL3

Interphase

Complete mitosis Mitotic defects

Per

cent

age

c

438

* ** **NS

100

80

60

40

20

0

siScr

am

siSCRAM

siKANSL1

siKANSL1

siKANSL3

siKANSL3

a

KANSL3

Cyclin A

CDK1

H3S10

Actin

KANSL1

Actin

150

10050

5037

1550

371

G1 S MG2

2 3 4

Figure 1 | KANSL1 and KANSL3 localize to spindle poles in mitosis and their depletion leads to mitotic defects. (a) NSL complex members KANSL1

and KANSL3 are expressed throughout the cell cycle in HeLa cells. Synchronization was confirmed by western blotting of various cell cycle markers,

as indicated. Molecular weight markers are indicated on the right and actin was used as a loading control. (b) Cells exhibit mitotic defects on knockdown

of NSL complex members KANSL1 and KANSL3. Representative still images from the live-cell analysis show dividing control (siSCRAMBLED) and KANSL1-

and KANSL3-silenced H2B-mCherry/a-tubulin-GFP HeLa cells. Typical phenotypes exhibited on knockdown of KANSL1 and KANSL3 include misaligned

chromosomes persistently attached to spindle poles, a prolonged metaphase delay and mitotic catastrophe (note membrane blebbing in the final panel).

Time in minutes is indicated in the upper left corners. Scale bars, 10mm. (c) Left side: quantification of the percentage of cells that complete mitosis over a

24-h time frame. All other cells remained arrested in prometaphase or had died by the end of the observation period. Right side: quantification of the

percentage of mitotic cells exhibiting the defects of misaligned chromosomes, multipolar spindles, lagging chromosomes or mitotic catastrophe. Error bars,

s.e.m. *Po0.05, **Po0.01, NS, non significant, according to unpaired one-tailed t-test. (d) Immunofluorescence of KANSL1 or KANSL3 proteins (green)

through the cell cycle. Tubulin is in red and DNA is in blue. Scale bars, 5 mm. KANSL1 and KANSL3 localize to spindle poles in mitosis.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8889 ARTICLE




In view of the previously described function for MCRS1(ref. 19), we decided to explore the function of KANSL1 andKANSL3 in the chromosomal pathway of microtubule assemblyin human cells. For this purpose, we performed a microtubuleregrowth assay, which can distinguish between the chromosomaland centrosomal pathways of microtubule nucleation in mitoticcells (see Methods)26. Immunofluorescence analysis on fixed cellsshowed that KANSL1 and KANSL3 localized to the centre ofchromosomal microtubule asters similar to MCRS1 (ref. 25). Inaddition, KANSL1 and KANSL3 also localized to centrosomalasters (Fig. 3a; Supplementary Fig. 2d). We then examinedwhether KANSL1 and KANSL3 were involved in chromosome-driven microtubule assembly. Quantification of the number ofmicrotubule asters in cells depleted of KANSL1, KANSL3 orMCRS1 during microtubule regrowth showed a statistically lowernumber than in control cells in line with our previous results onMCRS1 (on average 3 for MCRS1 or KANSL1 and 4 for KANSL3knockdown, versus 5 in the control; Fig. 3b)25. These resultsstrongly suggested that the KANSL proteins, in complex with

MCRS1 play an important role in chromosomal-dependentmicrotubule assembly. Consistently, co-silencing of MCAKtogether with KANSL1 or KANSL3 led to a partial rescue inthe number of asters in cells undergoing microtubule regrowth,again similarly to cells co-silenced for both MCAK and MCRS1(Supplementary Fig. 2e)25. Taken together, these results suggestthat KANSL1 and KANSL3 together with MCRS1 contribute tomicrotubule assembly in mitosis by stabilizing microtubules,likely by counteracting MT destabilizing activities.

One important property of MCRS1 is its specific localization toK-fibre microtubule minus ends25. We therefore examined thelocalizations of the KANSL proteins on K-fibres. HeLa cells wereincubated on ice to induce the depolymerization of the moredynamic spindle microtubules, leaving behind only K-fibres27.Immunofluorescence analysis showed that KANSL1 and KANSL3accumulated at K-fibre minus ends similarly to MCRS1 (Fig. 3c).This prompted us to look into the potential role of KANSL1 andKANSL3 in K-fibre dynamics. We first measured K-fibre lengthin KANSL1- and KANSL3-silenced cells and found that K-fibres

Con

trol

KA

NS

L1K

AN

SL3

–RanGTP +RanGTP+RanGTP,

+imp. β+RanGTP,

Δ-TPX2

–RanGTP

+RanGTP

+RanGTP +imp. β

+RanGTP, d-TPX2

Control KANSL1 KANSL3Per

cent

age

of b

eads

ass

ocia

ted

with

MT

s

0

10

20

30

40

50

Egg extractcontrol or Δ-TPX2

± RanGTP

Tubulin solution± importin-β

MT asters ??? (d)

WB (c)b

c

d

*

a

250

150

100

75

50

250

150

100

75

50

37

3M 1 2

KA

NS

L1K

AN

SL3

MO

F

3FLAG/HA-

– – –– + + ++ RanGTP

Control KANSL1 KANSL3 MOF

Input

α-Flag

α-TPX2

α-MCAK

1 2 3 4 5 6 7 8 9

*

Figure 2 | KANSL1 and KANSL3 promote microtubule assembly in a RanGTP-dependent manner in Xenopus egg extracts. (a) Coomassie blue-stained

gel of purified recombinant Drosophila KANSL1, KANSL3 and MOF tagged with 3FLAG/HA. (b) Schematic representation of the experimental design.

KANSL1 or KANSL3 was incubated in egg extract±RanGTP and retrieved on magnetic beads coupled with anti-HA antibodies. Beads were analysed by

western blot or incubated in pure tubulin to monitor microtubule assembly. (c) Western blots of KANSL1, KANSL3 and MOF beads retrieved from M-phase

Xenopus egg extracts in the presence (þ ) or absence (� ) of RanGTP. The control was performed in parallel with egg extract in the absence of recombinant

proteins. Anti-Flag antibodies were used to visualize KANSL1, KANSL3 and MOF. KANSL1 and KANSL3 interact with TPX2 and MCAK in a RanGTP-

dependent manner. (d) Microtubule assembly activity in pure tubulin. The KANSL beads retrieved from M-phase Xenopus egg extracts in the presence

(red bars) or absence (blue bars) of RanGTP were incubated in 20mM pure tubulin. Green bars correspond to beads retrieved from an extract containing

RanGTP and then incubated with importin-b and pure tubulin. Black bars represent beads retrieved from a xTPX2-depleted D-TPX2 egg extract containing

RanGTP. Beads were spun down on coverslips and processed for immunofluorescence to visualize microtubules. Representative images of beads are shown

on the left. Beads are autofluorescent. Scale bar, 5 mm. The graph on the right shows the percentage of beads interacting with microtubules in each

condition. KANSL1- and KANSL3-coated beads induce the assembly of microtubules when retrieved from an egg extract containing RanGTP. Data are from

three independent experiments. Error bars, s.d. *Po0.05 (unpaired t-test), NS, non significant.





were shorter than in control cells (Supplementary Fig. 2f). Wethen measured the interkinetochore distance in KANSL1- andKANSL3-silenced cells to test whether K-fibre dynamics wasaltered (Fig. 3d). We found that the distance was increasedcompared with controls. These data (Fig. 3d; SupplementaryFig. 2f) are consistent with what we described for MCRS1 (ref. 25)and reinforce the idea that KANSL1 and KANSL3 function incomplex with MCRS1 to control the rate of K-fibre microtubuledepolymerization at the minus end28. Taken together, weconclude that KANSL1 and KANSL3 interact and act togetherwith MCRS1 in mitosis, stabilizing chromosomal microtubuleand K-fibre minus ends.

KANSL3 is a microtubule minus-end binding protein. Tobetter understand the interaction between the KANSL proteins

and the mitotic microtubules, we first investigated theirmicrotubule-binding properties in vitro. Recombinant KANSL1and KANSL3 both co-pelleted with taxol-stabilized microtubulesin vitro, while another member of the NSL complex, MOF,did not (Supplementary Fig. 3a). Since KANSL proteins localizeto microtubule minus ends in mitosis (Fig. 1d and Fig. 3c), wethen examined whether they associated preferentially with theends of taxol-stabilized microtubules in vitro. As we showedpreviously, MCRS1 did not show any preferential end binding butassociated all along the microtubule lattice (Fig. 4a). By contrast,41% of microtubule-associated KANSL1 and 62% of microtubule-associated KANSL3 were indeed located at microtubuleends (Fig. 4a; Supplementary Fig. 3b–d). Moreover, they werepreferentially associated with only one of the two microtubuleends (in 86% of the cases for KANSL1 and 81% for KANSL3,Supplementary Fig. 3e).

MC

RS

1K

AN

SL

1

Tubulin

Tubulin

MCRS1

KANSL1 Merge

Merge

KA

NS

L3

MergeKANSL3Tubulin

Control MCRS1 KANSL1 KANSL3siRNA:

0

2

4

6

8

10


Num

ber

of M

T a

ster

s pe

r ce

ll ******

***

Tubulin

Tubulin MCRS1

KANSL1 Merge

Merge

Max. proj.

Max. proj.

MC

RS

1K

AN

SL1

KA

NS

L3

KANSL3Tubulin Merge Max. proj.


2

1

Inte

rkin

etoc

hore

dis

tanc

e (μ

m)

HEC HEC HECCREST CREST CREST CREST

DNA DNA DNA

Control MCRS1 KANSL1 KANSL3siRNA:HEC

DNA

******

***

a

b

c

d

Figure 3 | KANSL1 and KANSL3 are required for chromosomal microtubule assembly and K-fibre stabilization. (a) KANSL1 and KANSL3 localization to

chromosomal microtubule asters. Immunofluorescence images of mitotic HeLa cells fixed 5 min after nocodazole washout. Tubulin is shown in red; MCRS1,

KANSL1 and KANSL3 in green; and DNA in blue. KANSL1, KANSL3 and MCRS1 localize to the centre of microtubule asters. Scale bars, 5 mm. (b) KANSL1

and KANSL3 phenotypes in microtubule regrowth assay. Top: representative immunofluorescence images of microtubule asters in cells fixed 5 min after

nocodazole washout. Bottom: number of microtubule asters. Box and whisker plot: boxes show the upper and lower quartile, whiskers extend from the 10th

to the 90th percentile and dots correspond to outliers. n¼ 3. MCRS1-, KANSL1- and KANSL3-silenced cells had on average a lower number of microtubule

asters (3,429; 3,457 and 4,048, respectively), than control cells (4,996). More than 260 cells were analysed for each condition. ***P¼0.0001 (unpaired

t-test). (c) KANSL1 and KANSL3 localization to K-fibres. Cold-treated cells were fixed and stained with anti-tubulin (red) and anti-MCRS1, anti-KANSL1 or

anti-KANSL3 (green) antibodies. DNA is in blue. Single confocal slices are shown except for the images on the right that correspond as indicated to

maximum projections of 12 slices. KANSL1, KANSL3 and MCRS1 localize to the minus ends of K-fibres in mitosis. Scale bar, 5 mm. (d) Interkinetochore

distance. Control, MCRS1-, KANSL1- and KANSL3-silenced HeLa cells were fixed and stained for the outer-kinetochore marker Hec1 (green) and

CREST (red). The white arrows show paired sister kinetochores. The plot shows the interkinetochore distances in three independent experiments.

Box and whisker plot: boxes show the upper and lower quartile, whiskers extend from the 10th to the 90th percentile and dots correspond to outliers.

In MCRS1-, KANSL1- and KANSL3-silenced cells, the average interkinetochore distance is significantly increased (1,415; 1,504; 1,566mm, respectively,

versus 1,25 mm for the control cells). At least 221 kinetochores have been measured in each condition. Scale bar, 5 mm. ***P¼0,0001 (unpaired t-test).





The specific end binding of KANSL proteins was particularlyinteresting, as very few proteins are known to bind directly themicrotubule plus29 or minus end30–36. To determine which endof the microtubule KANSL3 preferentially associated with, weprepared taxol-stabilized, polarity-marked microtubules. In thesemicrotubules, the minus end is clearly marked by an intenseRhodamine tubulin ‘seed’37. Strikingly, we found that KANSL3preferentially associated with the microtubule minus ends in481% of the cases (Fig. 4b). Taken together, these data indicate

that KANSL3 is a novel direct microtubule minus-end-bindingprotein.

Since KANSL3 is capable of pulling down the entire NSLcomplex (Drosophila and mammalian) in in vitro reconstitutionassays (Fig. 4c), we decided to test whether KANSL3 recruitsMCRS1 to the microtubule minus ends in mitotic cells. In theabsence of KANSL3, although MCRS1 levels are unaffected(Supplementary Fig. 1c), its localization to the spindle poles wasindeed strongly reduced (Fig. 4d; Supplementary Fig. 4a).

bα-Tubulin α-HA Merge

KA

NS

L1K

AN

SL3

MC

RS

1C

ontr

ol

α-MBPα-Tubulin Merge

TubulinKANSL1

TubulinHA

TubulinKANSL3

TubulinMCRS1

a

KA

NS

L3

+ −MT

Seed

KANSL3

0

20

40

60

80

100

KA

NS

L3 lo

caliz

atio

non

MT

end

s

Minus end Plus end

Tubulin MCRS1 Merge

siR

NA

cont

rol

siR

NA

KA

NS

L3

TubulinMCRS1

DNA

c

NSL1MBD-R2NSL3MOF

MCRS2NSL2

WDS

250

150

100

75

50

37

M M1 2

KANSL1/PHF20KANSL3

KANSL2

MCRS1/hMOF

WDR5

250150

100

75

50

37

3FLAG-dKANSL3 3FLAG-KANSL3 d

NSL complex

KANSL3

KANSL1

MCRS1

Microtubule (MT)

Interphase

KANSL3KANSL1

Mitosis

+ − Chromatin

e

%

Figure 4 | KANSL3 is a microtubule minus-end-binding protein. (a) KANSL1 and KANSL3 localization to microtubule ends in vitro. Taxol-stabilized

microtubules were incubated with recombinant KANSL1, KANSL3, xMCRS1 or buffer (control), as indicated. Microtubules were spun down on coverslips

and processed for immunofluorescence with anti-tubulin (red) and either anti-HA (for KANSL proteins) or anti-MBP (for MCRS1) antibodies (green).

Arrows indicate KANSL1 and KANSL3 accumulation at microtubule ends. Scale bar, 5 mm. (b) KANSL3 binds to the minus end of polarity-marked

microtubules in vitro. Polarity-marked microtubules were incubated with KANSL3, spun down on coverslips and processed for immunofluorescence.

The bright microtubule seed at the minus end is in green, tubulin is in blue and KANSL3 in red, as shown in the drawing. KANSL3 associates with the

microtubule minus end. Bottom: quantification of the microtubule minus end localization of KANSL3. The percentage of plus-end and minus-end signal for

KANSL3 is shown. KANSL3 binds preferentially to minus ends (81.3%) versus plus ends (18.7%). n¼ 3. More than 250 microtubules were analysed. Error

bars, s.d. (c) KANSL3 nucleates a complete NSL complex in vitro. All seven members of the NSL complex were expressed in SF21 cells as untagged proteins

except KANSL3 that was tagged with 3FLAG. Flag pull-down from the cellular extract retrieved a complete heptameric NSL complex. This indicates that

KANSL3 is a structurally central component of the NSL complex, both in the context of the Drosophila and human proteins. (d) KANSL3 silencing in HeLa

cells results in the loss of MCRS1 localization to spindle poles. Representative immunofluorescence pictures of control or KANSL3-silenced HeLa cells, as

indicated. Tubulin is in red, MCRS1 in green and DNA in blue. Scale bar, 5 mm. (e) Summary. In interphase, KANSL1 and KANSL3 proteins are chromatin

bound and regulate expression of housekeeping genes. During mitosis, these proteins relocate to the mitotic spindle and are important for cell division.

Moreover, KANSL3 binds directly to microtubule minus ends in vitro and localizes to K-fibre microtubule minus ends in the dividing cell. Thus, KANSL

proteins are able to adopt distinct tasks in different phases of cell cycle to ensure cellular homeostasis.





This was specific for MCRS1 as neither TPX2 nor NuMAlocalizations changed on KANSL3 silencing (SupplementaryFig. 4b,c). These data suggest that KANSL3 directly binds tothe K-fibre microtubule minus ends and recruits MCRS1. Takentogether, our results show that KANSL1, KANSL3 and MCRS1constitute a mitosis-specific microtubule minus-end-associatedcomplex essential for chromosomal microtubule assembly andcorrect K-fibre dynamics (Fig. 4e).

DiscussionEarlier studies have considered the function of KANSL proteinsexclusively in epigenetic regulation in the nucleus. Here we showthat the NSL complex possesses a novel microtubule-bindingand stabilizing activity in mitosis distinct from its interphasetranscriptional activation activities. These functions are under thecontrol of the RanGTP pathway, which allows the recycling ofnuclear interphase complexes as microtubule regulators inmitosis. We describe a novel contribution of KANSL1 andKANSL3 to spindle assembly. Knockdown of KANSL proteinsleads to marked and terminal mitotic defects, highlighting theessential nature of the KANSL proteins to cells. We show thatKANSL1 and KANSL3 are able to regulate microtubule stabilityboth in cells and in vitro. Our data strongly point to the existenceof a functional microtubule-binding subcomplex consisting of atleast KANSL1, KANSL3 and MCRS1 in mitotic cells. Although afew chromatin-binding factors have been shown to play a role inspindle assembly18, we show here for the first time that achromatin-binding complex maintained in interphase andmitosis plays two distinct functions in these different cell cyclestages. Future work will determine whether the full NSL complexis involved in this mitotic function, or whether there are differentspecific subcomplexes. It is interesting to speculate that the cellmay possess a large cytoplasmic pool of the NSL complex, likelystored in preparation for resumption of transcription in G1.

We also report the discovery of a new autonomousmicrotubule minus-end-binding protein, KANSL3. Thus far,only one protein family has recently been described to possessthis property (namely Patronin/CAMSAPs)30,32,36,38–40. SinceKANSL3 is predominantly nuclear in interphase, it may onlyassociate with microtubules during mitosis. This makes KANSL3a new microtubule minus-end-binding protein with a rolein mitosis. Future work should help us to elucidate thestructural elements that enable KANSL3 to recognizemicrotubule minus ends.

We propose that KANSL proteins play an important role incellular homeostasis during different stages of the cell cycle. Onthe one side, they are transcriptional regulators targetingpromoters of essential housekeeping genes during interphaseand on the other side by stabilizing microtubules, KANSLproteins ensure faithful segregation of the genome during mitosis.Distinct contribution of these proteins during interphase andmitosis could also explain why cells as well as organisms (flies andhumans) are sensitive to the dosage of KANSL proteins and theirreduction or loss is not tolerated and is associated withmicrodeletion syndrome or cancer.

MethodsCell culture. HeLa and HEK293 Flp-In Trex (Life Technologies) cells were grownin DMEM supplemented with Glutamax, 9% heat-inactivated fetal calf serum and1� penicillin and streptomycin (Life Technologies). The stable HeLa Kyoto cellline expressing GFP-tubulin and H2B-mCherry20 was maintained in the presenceof 2 mg ml� 1 puromycin (Carl Roth GmbH) and 500 mg ml� 1 G418 (Sigma) untiltransfection. The stable HeLa cell line expressing GFP-centrin41 was maintained inthe presence of 500 mg ml� 1 G418 (Sigma).

A stable HEK293 cell line expressing FLAG-MCRS1 was generated bycotransfection of the pFlag-MCRS1 construct25 and the pBABE-puro vector

(Addgene). Clones were selected using puromycin (2.5 mg ml� 1; Sigma-Aldrich)and selected for FLAG-MCRS1 expression by western blot.

Stable inducible HEK293 Flp-In Trex (Life Technologies) cell lines carryingKANSL1-FBH (3XFLAG/biotin acceptor site/6XHis), KANSL3-FBH andMCRS1-FBH were generated by flippase recognition target (FRT) recombinationaccording to the product manual. These cell lines were maintained in the presenceof 15mg ml� 1 blasticidin and 150mg ml� 1 hygromycin. The day beforesynchronization, cell lines were induced by addition of 100 ng ml� 1 doxycycline.

Cell synchronization. For HeLa cell synchronization, cells were plated at 20%confluence and incubated with 3 mM thymidine (Sigma-Aldrich). After 16 h, cellswere released into fresh medium for 8 h. Cells were then incubated with 3 mMthymidine for 14 h. For the ‘S-phase’ sample, these cells were released into freshmedium for 2 h and then harvested by trypsinization. Part of the sample wasreserved for fluorescence-activated cell sorting (FACS) and the other lysed in RIPAbuffer (phosphate-buffered saline (PBS) with 1% NP-40, 0.5% sodium deox-ycholate, 0.1% sodium dodecyl sulphate (SDS)), vortexed for 10 s and clarified bycentrifugation at 10,000 r.p.m. for 15 min. For the ‘G2-phase’ and ‘M-phase’samples, cells were released into fresh medium for 6 h and then incubated with9 mM RO3306 (Alexis Biochemicals) or 50 ng ml� 1 nocodazole (Calbiochem) for8 h. G2-phase samples were harvested by trypsinization. M-phase samples wereharvested by shake-off. For the G1 sample, cells were released into fresh mediumfor 8 h and subsequently incubated in 20 mM lovastatin (Santa Cruz) for 8 h. Thecells were harvested by trypsinization.

HEK293 Flp-In Trex cells were synchronized in mitosis by incubation for 14 hwith 10mM (þ )-S-trityl-L-cysteine (STLC, Alfa Aesar)42.

FACS. Cells were washed in PBS once and then fixed in cold 75% ethanol withrotation overnight. Cells were stored at 4 �C until needed. For analysis, cells werestained with propidium iodide, treated with 0.2 mg ml� 1 RNase A and observed ona BD LSRII instrument. Around 10,000 to 15,000 cells were measured percondition.

Antibodies. Immunofluorescence and immunoprecipitations of KANSL1and KANSL3 were performed using antibodies previously described3.Immunofluorescence for MCRS1 was performed using an antibody previouslydescribed25.

The following primary antibodies were used for immunofluorescence: a-tubulinDM1A (Sigma T9026; 1:1,000), a/b-tubulin (Abcam ab44928; 1:1,000), a-tubulin(Merck Millipore 04-1117; 1:400) and HA (Covance MMS-101P; 1:100). The MCAKpolyclonal antibodies were a gift from E. Karsenti lab; the polyclonal Hec1 (9G3.2GeneTex) antibody was used at 1:100 for immunofluorescence; CREST antibodieswere used at 1:100 (Antibodies Incorporated 15-235-F); and the polyclonal MBP,hTPX2, NuMA and xTPX2 antibodies were produced in the Vernos lab.

The following primary antibodies were used for detectionby western blot. All antibodies were used at a 1:1,000 dilution except whereotherwise stated. KANSL1 (Abnova corporation PAB20355), KANSL3 (Sigma-Aldrich HPA035018), GAPDH (Bethyl Laboratories A300-641A, 1:6,000), Rad21(Merck Millipore 05-908), Cyclin A (Abcam ab38), CDK1 (Abcam ab18), H3S10(Abcam ab5176; 1:10,000), beta-actin (CST #4967; 1:3000), DM1A for a-tubulin(Sigma T9026; 1:10,000) and M2 for FLAG tag (Sigma F3165). The polyclonalhTPX2 antibody was produced in the Vernos lab.

Secondary antibodies (anti-mouse, anti-rat, anti-rabbit or anti-human)conjugated to Alexa488, 555, 568, 680 or 647 (Molecular Probes) were used at1:500–1:1,000 for immunofluorescence and 1:10,000 for western blot.

Co-immunoprecipitation in HeLa and HEK293 cells. Cells were harvested eitherby shake-off (for M-phase samples) or trypsinization (for unsynchronizedsamples). The contents of a 15-cm tissue culture plate were resuspended in 2 ml ofhigh Tween20 HKMGT buffer (25 mM HEPES pH 7.6, 150 mM KCl, 12.5 mMMgCl2, 10% glycerol, 0.4% Tween20) with Complete protease inhibitors (Roche)added. The sample was sonicated for a total of 50 pulses output 3 duty cycle 30%on a Branson sonifier 250 with a microtip and subsequently clarified by spinningat 15,000g for 15 min at 4 �C. The cleared lysate was incubated either withimmunoprecipitating antibody or directly with 30 ml pre-washed FLAG beads(Sigma) overnight. For endogenous co-IP, cleared lysates were incubated overnightwith 5ul of antibody (see above) and then 1 h with pre-washed mixed ProtA and Gbeads. Endogenous co-IPs were washed three times in the lysis buffer. HEK FLAGIPs were washed twice in a low Tween, low stringency buffer (25 mM HEPES pH7.6, 75 mM KCl, 12.5 mM MgCl2, 10% glycerol, 0.1% Tween20).

Western blot analysis. Western blots in Fig. 1 and Supplementary Figs 1 and 2were performed as follows. Following transfer, the 0.45 mm polyvinylidenedifluoride membrane (Roche) was blocked for 1 h at room temperature in 5%bovine serum albumin (BSA) in PBS with 0.05% Tween20 (Sigma). Primaryantibodies were incubated in 2% BSA, 0.05% Tween20 in PBS at 4 �C overnight.Secondary antibodies conjugated with horseradish peroxidase (GE Healthcare)were incubated in the same buffer at room temperature for 1 h.





Western blots in Fig. 2 and Supplementary Fig. 3 were performed using 0.45 mmnitrocellulose membrane (Whatman). Blocking and antibody incubations wereperformed using PBS with 3% milk and 0.1% Tween20 (Serva). Primary andsecondary antibodies were incubated 1 h at room temperature. Secondaryantibodies conjugated with AlexaFluors (see above) were used.

Uncropped western blots are presented in Supplementary Fig. 5.

RNA interference. Cells were seeded at 15,000 cells per six well the day beforetransfection. Three-hundred picomoles of siRNA and 15 ml of XtremeGene siRNAtransfection reagent (Roche) were used per six well (and scaled appropriately forsmaller volumes). Transfection was performed as per supplier protocol. siRNAduplexes were ordered from MWG Operon using the following sequences (a mix ofsequences 1þ 2 was prepared for transfection):

KANSL1 (1): 50-CGGCAACGCCAACAUCCUU-30

KANSL1 (2): 50-GAAGCGGAGGCUUGUUCGA-30

KANSL3 (1): 50-GGCACGCAGCGTGATGAAT-30

KANSL3 (2): 50-GGAUGCUUGUGUCAUCCAA-30

MCRS1: 50-GGCAUGAGCUCUCCGGAC-30

MCAK: 50-GAUCCAACGCAGUAAUGGU-30

MCRS1 and MCAK siRNAs were previously validated25.

Immunofluorescence. For KANSL1 and KANSL3 immunofluorescence in Fig. 1d,Supplementary Figs 1f and 3d and KANSL3 immunofluorescence in Fig. 3, cellswere grown on precision no. 1.5 coverslips (Carl Roth LH24.1) in six-well plates.Cells were pre-lysed for 20 s in PHEM (60 mM PIPES, 25 mM HEPES, 10 mMEGTA and 2 mM MgCl2) buffer with 0.5% Triton X100, washed once in PHEMbuffer and fixed in 3.7% formaldehyde (Sigma F8775) in PHEM buffer with 0.1%Triton X100 at room temperature for 8 min. Cells were blocked in 2% BSA inPHEM þ 0.1% Triton X100 for 30 min at room temperature. Primary antibodieswere incubated in blocking buffer overnight at 4 �C. Secondary antibodies con-jugated with AlexaFluors (see ‘Antibodies’ above) were diluted 1:500 in blockingbuffer and incubated at room temperature for 1 h. Where appropriate, Hoechst33342 (Life technologies) was added in the secondary antibody mix to stain DNA.Coverslips were mounted using Fluoromount G (Southern Biotech).

These samples were visualized with a 63� objective on a Zeiss Observer.Z1with the Yokogawa CSU-X1 spinning disk head and the Zeiss Axiocam MRmcamera, with the exception of Fig. 1d KANSL3 immunofluorescence, which wasvisualized on the Zeiss LSM780 confocal microscope.

For KANSL1 immunofluorescence in Fig. 3 and all other immunofluorescencesin Figs 3 and 4 and Supplementary Figs 2 and 4, cells were grown on coverslips insix-well plates and fixed in � 20 �C methanol for 10 min. Blocking and antibodydilution buffer were 0.5% BSA (Sigma) and 0.1% Triton X100 (Sigma) in PBS.Primary and secondary antibodies were incubated for 1 h at room temperature.Coverslips were mounted in 10% Mowiol (Calbiochem) in 0.1 M Tris-HCl at pH8.2, 25% glycerol (Merck).

For immunofluorescences in Figs 2 and 4a/b, samples were spun down at 1,000gfor 10 min on coverslips before fixation in � 20 �C methanol for 10 min.

These samples were visualized with a 63� objective on an inverted DM1-6000Leica widefield fluorescence microscope. Pictures were acquired with the LeicaApplication Suite software. Images were processed with ImageJ and Photoshop(Adobe) and assembled as figures using Illustrator (Adobe). Line scans forfluorescence intensity quantification along the spindles were performed usingImageJ.

Live-cell imaging. Cells were seeded and treated in eight-well coverslip-bottomeddishes (ibidi GmbH cat. no 80826). Two days following transfection, dishes weretransferred to a temperature- and CO2-controlled Tokai Hit stage incubation unit.Samples were visualized using a Zeiss Observer.Z1 with the CSU-X1 spinning diskhead (Yokogawa) and the QuantEM:512SC camera (Photometrics). Z-stacks ofcells were taken at 3-min intervals using a water immersion 63� objective. Forquantifications additional videos were taken at 8-min intervals using a 40� longworking distance objective. Time lapse was performed for a 24-h duration. Laserpower and exposure times were kept to an absolute minimum.

Recombinant proteins. Recombinant proteins were purified either fromEscherichia coli or SF21 cells.

RanQ69L and x-importin-b were tagged with His-tag, produced in E. coli andpurified using Ni-NTA agarose beads (Qiagen). RanQ69L was then loaded with1 mM GTP in PBS 2 h at 4 �C. MBP-tagged x-MCRS1 was produced in E. coli andpurified25 using amylose resin (NEB) according to the manufacturer’srecommendations.

Recombinant full-length Drosophila melanogaster KANSL1, KANSL3 andMOF were expressed with an N-terminal 3FLAG/HA tag using the Bac-to-Bac(Invitrogen) system in SF21 cells. Fifty millilitres of infected cells were harvested bycentrifugation, washed in PBS and resuspended in high Tween20 HKMGT buffer(25 mM HEPES pH 7.6, 150 mM KCl, 12.5 mM MgCl2, 10% glycerol, 0.4%Tween20) with Complete protease inhibitors (Roche) added. Following three freezethaw cycles between liquid nitrogen and a 20 �C water bath, the lysate wasultracentrifuged at 100,000g for 30 min at 4 �C. The cleared lysate was incubated

with 80 ml pre-washed FLAG beads (Sigma) overnight. Following one wash in theabove HKMGT buffer and three in a low Tween20 (0.1%) HKMGT buffer, thesamples were eluted using 250mg ml� 1 of 3XFLAG peptide (Sigma) in lowTween20 HKMGT with protease inhibitors added. Proteins were quantified byCoomassie blue staining and snap frozen in aliquots.

Beads experiments in Xenopus egg extracts. Fresh cytosolic factor (CSF)-arrested Xenopus EE was prepared as previously described43. xTPX2 depletionfrom Xenopus egg extracts was performed by two 30-min rounds of depletion usinghomemade anti-xTPX2 antibodies coupled with Protein A dynabeads (LifeTechnologies). Recombinant KANSL1, KANSL3, MOF and xMCRS1 wereincubated in CSF-egg extract at 100 nM final concentration for 30 min at 20 �Cwith or without addition of 10 mM RanGTPQ69L. Protein A-coated Dynabeads(Invitrogen) were washed three times in PBS 0.1% Triton X100 (Sigma) andincubated with the indicated antibodies (anti-HA for KANSL1, KANSL3 and MOF;anti-MBP for xMCRS1) diluted in the same buffer for 1 h at room temperature,following the manufacturer’s indications. Beads were retrieved on a magnet andwashed twice in PBS 0.1% Triton X100 and twice in CSF-XB (10 mM HEPES;100 mM KCl; 0,1 mM CaCl2, 2 mM MgCl2; 50 mM sucrose; 5 mM EGTA). Beadswere then incubated in CSF-egg extract (at 1:3) for 1 h at 4 �C. Beads wererecovered on a magnet, washed twice in CSF-XB and twice in BRB80 (80 mMPIPES; 1 mM MgCl2; 5 mM EGTA; pH 6.8) and resuspended in BRB80. They wereeventually incubated in the presence of 5 mM importin-b. Two microlitres of beadswas added to 30 ml of pure tubulin (20 mM tubulin purified from calf brain, BRB80,1 mM GTP) and incubated at 37 �C for 10 min. The sample was fixed in 500 ml 1%glutaraldehyde in BRB80, and centrifuged onto a coverslip through a 10% glycerolcushion (vol/vol in BRB80) at 1,500g for 10 min. Coverslips were postfixed for10 min in � 20 �C methanol before processing for immunofluorescence44.

To evaluate microtubule efficiency around the beads, the number of beadsassociated with a microtubule was quantified. At least 300 beads were counted ineach condition. For the western blot analysis in Fig. 2c, all IPs were performed atleast three times.

Stable isotope labelling of amino acids in cell culture. HEK293 cells stablyexpressing FLAG-MCRS1 were grown in SILAC DMEM R6K8 medium (Dundeecell products); control HEK293 cells were grown in SILAC DMEM R0K0 medium(Dundee cell products), both media were supplemented with 2 mM L-glutamine(Invitrogen), 10% fetal bovine serum (Dundee cell products) and 1� penicillinand streptomycin (Invitrogen). Cells were grown for 10 days with 5% CO2 in ahumid atmosphere on a poly-D-lysine layer (Sigma) with media changed every48 h. Cells were synchronized over a 48-h period using first a 16-h incubation with2 mM thymidine (Sigma), release into fresh medium for 8 h, arrest with 2 mMnocodazole (Sigma) for 16 h, followed by a 30-min release. Cells were collected byshake-off. Cell pellets were resuspended in extraction buffer (20 mM HEPES pH7.8; 175 mM NaCl; 2,5 mM MgCl2; 10% glycerol; 1 mM dithiothreitol (DTT;Sigma); 1 mM phenylmethylsulphonyl fluoride (Sigma); protease inhibitor cocktail(Roche); 2 mM okadaic acid (Sigma); 1 mM orthovanadate (Sigma); 40 mMglycerophosphate (Sigma); and 50 mM NaF (Sigma)). Cells were lysed using anitrogen bomb at 1,500 p.s.i. for 15 min.

An equal amount of protein (51.15 mg) of control and tagged protein wasmixed and a FLAG pull-down was carried out using an ANTI-FLAG M2 affinitygel (Sigma).

The protein extract was incubated with the ANTI-FLAG M2 affinity gel for 2 hat 4 �C and washed four times with 50 mM Tris pH 7.4; 150 mM NaCl; 1 mMEDTA and 1% IGEPAL CA-630 (Sigma). FLAG proteins were eluted from theresin by competition with 400mg ml� 1 of FLAG peptide (Sigma). FLAG proteinswere concentrated in a speedVac, separated by SDS–polyacrylamide gelelectrophoresis and visualized using the NOVEX colloidal blue staining kit(Invitrogen). Bands were cut out and analysed by mass spectrometry at theCentre for Genomic Regulation proteomics facility.

Microtubule regrowth and cold-stable assays. For microtubule regrowth assays,3 mM nocodazole (Sigma) was added to the cell medium for 3 h, and washed outthree times in PBS and once in medium at 37 �C. Cells were fixed at 5 min afternocodazole washout, and processed for immunofluorescence as described. Resultswere quantified counting the number of microtubule asters in at least 100 cells toobtain the average per cell.

For K-fibre length assays, cells were incubated on ice for 10 min in L15 medium(Sigma) supplemented with 20 mM HEPES (Sigma) at pH 7.3 and washed oncewith cold PBS before fixation. To measure K-fibre length, cells were treated with2 mM STLC (Sigma) for 3 h and then subjected to the cold treatment in thepresence of STLC. K-fibres of at least 40 monopolar spindles in each conditionwere measured using ImageJ.

Microtubule co-pelleting experiment. Taxol microtubules were preparedincubating 20mM of bovine tubulin at 37 �C in BRB80þ 1 mM GTP. After5-min incubation, taxol was then progressively added at the following finalconcentrations: 0.2, 2 and 20 mM.





Three-micromolar taxol-stabilized microtubules were incubated with 250 nM ofrecombinant KANSL1, KANSL3 or Drosophila MOF, as indicated, for 30 min at4 �C. Microtubules were then pelleted through a 30% glycerol cushion in 5 mMtaxol in BRB80 at 100,000g in a TLA-100 rotor (Beckman-Coulter) for 24 min at22 �C. Supernatant and pellet fractions were separated by SDS–polyacrylamide gelelectrophoresis and subjected to western blot analysis.

In vitro experiments with taxol-stabilized microtubules. For localizationstudies, microtubules stabilized with 1.5 mM taxol were incubated for 10 min atroom temperature with 100 nM MBP-xMCRS1, KANSL1 or KANSL3, as indicated,in BRB80. Samples were then fixed with 1% glutaraldehyde, 5 mM taxol in BRB80and pelleted on coverslips through a 25% glycerol, BRB80 cushion at 4,000g for10 min at room temperature. Coverslips were then recovered and fixed for 10 minin � 20 �C methanol and processed for immunofluorescence.

In vitro experiments with polarity-marked microtubules. Polarity-markedmicrotubules were prepared from microtubule bright seeds. The seeds wereprepared by mixing 1:1 Rhodamine–tubulin, unlabelled tubulin in BRB80, 1 mMGMPCPP (Jena Biosciences), 1 mM DTT and incubated 10 min on ice beforecentrifugation at 100,000g for 10 min at 4 �C. Polymerization was performed byincubation 25 min at 37 �C. Polymerized seeds were then diluted five times with20mM unlabelled tubulin, 1 mM GTP, 1 mM DTT in BRB80 and incubated at37 �C for 20 min. The polarity-marked microtubules were then stabilized adding10mM taxol and used for in vitro visualization experiments.

KANSL complex reconstitution in vitro. Two baculovirus constructs were createdin which all seven members of the KANSL complex were overexpressed underalternating viral p10 and pH promoters. KANSL1, KANSL2, MOF, MCRS1/2,PHF20/MBD-R2 and WDR5/WDS were in their native state (untagged). KANSL3was tagged with 3FLAG to enable it to function as the bait. SF21 cells co-infectedwith the two viruses produce high levels of all the protein members simultaneously.After incubating the extract (see ‘Recombinant proteins’ for preparation) from cellsinfected with these viruses with FLAG beads, a complete KANSL complex waspurified. This was performed individually for both human and Drosophilacomplexes.

References1. Cai, Y. et al. Subunit composition and substrate specificity of a

MOF-containing histone acetyltransferase distinct from the male-specific lethal(MSL) complex. J. Biol. Chem. 285, 4268–4272 (2010).

2. Dias, J. et al. Structural analysis of the KANSL1/WDR5/KANSL2 complexreveals that WDR5 is required for efficient assembly and chromatin targeting ofthe NSL complex. Genes Dev. 28, 929–942 (2014).

3. Mendjan, S. et al. Nuclear pore components are involved in the transcriptionalregulation of dosage compensation in Drosophila. Mol. Cell 21, 811–823(2006).

4. Feller, C. et al. The MOF-containing NSL complex associates globally withhousekeeping genes, but activates only a defined subset. Nucleic Acids Res. 40,1509–1522 (2012).

5. Lam, K. C. et al. The NSL complex regulates housekeeping genes in Drosophila.PLoS Genet. 8, e1002736 (2012).

6. Raja, S. J. et al. The nonspecific lethal complex is a transcriptional regulator inDrosophila. Mol. Cell 38, 827–841 (2010).

7. Chelmicki, T. et al. MOF-associated complexes ensure stem cell identity andXist repression. Elife 3, e02024 (2014).

8. Ravens, S. et al. MOF-associated complexes have overlapping and unique rolesin regulating pluripotency in embryonic stem cells and during differentiation.Elife, e02104 (2014).

9. Koolen, D. A. & de Vries, B. B. A. KANSL1-Related Intellectual DisabilitySyndrome. (eds Pagon, R. A. et al.) (GeneReviews, 1993).

10. Koolen, D. A. et al. Mutations in the chromatin modifier gene KANSL1 causethe 17q21.31 microdeletion syndrome. Nat. Genet. 44, 639–641 (2012).

11. Zollino, M. et al. Mutations in KANSL1 cause the 17q21.31 microdeletionsyndrome phenotype. Nat. Genet. 44, 636–638 (2012).

12. Gilissen, C. et al. Genome sequencing identifies major causes of severeintellectual disability. Nature 511, 344–347 (2014).

13. Gottesfeld, J. M. & Forbes, D. J. Mitotic repression of the transcriptionalmachinery. Trends Biochem. Sci. 22, 197–202 (1997).

14. Kruhlak, M. J. et al. Regulation of global acetylation in mitosis through loss ofhistone acetyltransferases and deacetylases from chromatin. J. Biol. Chem. 276,38307–38319 (2001).

15. Karsenti, E. & Vernos, I. The mitotic spindle: a self-made machine. Science 294,543–547 (2001).

16. Kalab, P., Weis, K. & Heald, R. Visualization of a Ran-GTP gradient ininterphase and mitotic Xenopus egg extracts. Science 295, 2452–2456 (2002).

17. Meunier, S. & Vernos, I. Microtubule assembly during mitosis - from distinctorigins to distinct functions? J. Cell Sci. 125, 2805–2814 (2012).

18. Yokoyama, H & Gruss, O. J. New mitotic regulators released from chromatin.Front. Oncol. 3, 308 (2013).

19. Yokoyama, H., Rybina, S., Santarella-Mellwig, R., Mattaj, I. W. & Karsenti, E.ISWI is a RanGTP-dependent MAP required for chromosome segregation.J. Cell Biol. 187, 813–829 (2009).

20. Neumann, B. et al. Phenotypic profiling of the human genome by time-lapsemicroscopy reveals cell division genes. Nature 464, 721–727 (2010).

21. Masui, Y. & Clarke, H. J. Oocyte maturation. Int. Rev. Cytol. 57, 185–282(1979).

22. Wuhr, M. et al. Deep proteomics of the Xenopus laevis egg using anmRNA-derived reference database. Curr. Biol. 24, 1467–1475 (2014).

23. Scrofani, J., Sardon, T., Meunier, S. & Vernos, I. Microtubule nucleation inmitosis by a RanGTP-dependent protein complex. Curr. Biol. 25, 131–140(2015).

24. Schatz, C. A. et al. Importin alpha-regulated nucleation of microtubules byTPX2. EMBO J. 22, 2060–2070 (2003).

25. Meunier, S. & Vernos, I. K-fibre minus ends are stabilized by a RanGTP-dependent mechanism essential for functional spindle assembly. Nat. Cell Biol.13, 1406–1414 (2011).

26. Tulu, U. S., Fagerstrom, C., Ferenz, N. P. & Wadsworth, P. Molecularrequirements for kinetochore-associated microtubule formation in mammaliancells. Curr. Biol. 16, 536–541 (2006).

27. Rieder, C. L. & Borisy, G. G. The attachment of kinetochores to the pro-metaphase spindle in ptk1 cells - recovery from low-temperature treatment.Chromosoma 82, 693–716 (1981).

28. Maresca, T. J. & Salmon, E. D. Intrakinetochore stretch is associated withchanges in kinetochore phosphorylation and spindle assembly checkpointactivity. J. Cell Biol. 184, 373–381 (2009).

29. Galjart, N. Plus-end-tracking proteins and their interactions at microtubuleends. Curr. Biol. 20, R528–R537 (2010).

30. Tanaka, N., Meng, W., Nagae, S. & Takeichi, M. Nezha/CAMSAP3 andCAMSAP2 cooperate in epithelial-specific organization of noncentrosomalmicrotubules. Proc. Natl Acad. Sci. USA 109, 20029–20034 (2012).

31. Richardson, C. E. et al. PTRN-1, a microtubule minus end-binding CAMSAPhomolog, promotes microtubule function in Caenorhabditis elegans neurons.Elife 3, e01498 (2014).

32. Meng, W., Mushika, Y., Ichii, T. & Takeichi, M. Anchorage of microtubuleminus ends to adherens junctions regulates epithelial cell-cell contacts. Cell135, 948–959 (2008).

33. Marcette, J. D., Chen, J. J. & Nonet, M. L. The Caenorhabditis elegansmicrotubule minus-end binding homolog PTRN-1 stabilizes synapses andneurites. Elife 3, e01637 (2014).

34. Goshima, G. et al. Genes required for mitotic spindle assembly in DrosophilaS2 cells. Science 316, 417–421 (2007).

35. Nagae, S., Meng, W. & Takeichi, M. Non-centrosomal microtubules regulateF-actin organization through the suppression of GEF-H1 activity. Genes Cells18, 387–396 (2013).

36. Goodwin, S. S. & Vale, R. D. Patronin regulates the microtubule network byprotecting microtubule minus ends. Cell 143, 263–274 (2010).

37. Hyman, A. A. Preparation of marked microtubules for the assay of the polarityof microtubule-based motors by fluorescence. J. Cell Sci. Suppl. 14, 125–127(1991).

38. Hendershott, M. C. & Vale, R. D. Regulation of microtubule minus-enddynamics by CAMSAPs and Patronin. Proc. Natl Acad. Sci. USA 111,5860–5865 (2014).

39. Wang, H., Brust-Mascher, I., Civelekoglu-Scholey, G. & Scholey, J. M. Patroninmediates a switch from kinesin-13-dependent poleward flux to anaphase Bspindle elongation. J. Cell Biol. 203, 35–46 (2013).

40. Jiang, K. et al. Microtubule minus-end stabilization by polymerization-drivenCAMSAP deposition. Dev. Cell 28, 295–309 (2014).

41. Piel, M., Meyer, P., Khodjakov, A., Rieder, C. L. & Bornens, M. Therespective contributions of the mother and daughter centrioles tocentrosome activity and behavior in vertebrate cells. J. Cell Biol. 149, 317–330(2000).

42. Dunsch, A. K., Linnane, E., Barr, F. A. & Gruneberg, U. The astrin-kinastrin/SKAP complex localizes to microtubule plus ends and facilitates chromosomealignment. J. Cell Biol. 192, 959–968 (2011).

43. Sardon, T., Peset, I., Petrova, B. & Vernos, I. Dissecting the role of Aurora Aduring spindle assembly. EMBO J. 27, 2567–2579 (2008).

44. Peset, I. et al. Function and regulation of Maskin, a TACC familyprotein, in microtubule growth during mitosis. J. Cell Biol. 170, 1057–1066(2005).

AcknowledgementsWe are grateful to Dr Jan Ellenberg (EMBL Heidelberg) for his gift of GFP-tubulin /H2B-mCherry HeLa Kyoto line, Dr Alexey Khodjakov (Wadsworth Center) for his gift ofthe GFP-centrin HeLa line and Dr Huseyin Besir (EMBL Heidelberg) for SF21 cells.We would like to thank Andreas Wurch and Petra Kindle from the MPIIE FACS and





Imaging Facilities, respectively, for their advice and practical assistance. We thank theCentre for Genomic Regulation proteomics facility and Nuria Mallol and Jacobo Cela forexcellent technical assistance. M.S. and A.A. would like to thank Tomacz Chelmicki forpioneering work in this project. This work was supported by DFG funded CRC746,CRC992 and CRC 1140 awarded to A.A. A.A. is part of the EU funded Network ofExcellence (EpiGeneSys). IV lab was supported by the Spanish Ministry of Economy andCompetitiveness grants BFU2012-37163.

Author contributionsS.M. performed all the experiments in Xenopus egg extracts and the in vitro assays in thepresence of microtubules. M.S. performed live-cell imaging and analysis. S.M. andM.S. performed the immunofluorescence-based assays and quantifications. Cloningand virus generation for recombinant proteins was performed by N.V.N. Singlerecombinant proteins were expressed and purified by M.S. The in vitro reconstitution ofthe KANSL complexes was performed by N.V.N. L.A. performed the SILAC experiment.A.A., I.V., S.M. and M.S. designed the experiments, analysed the data and prepared themanuscript.

Additional informationSupplementary Information accompanies this paper at http://www.nature.com/naturecommunications

Competing financial interests: The authors declare no competing financial interests.

Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/

How to cite this article: Meunier, S. et al. An epigenetic regulator emerges asmicrotubule minus-end binding and stabilizing factor in mitosis. Nat. Commun. 6:7889doi: 10.1038/ncomms8889 (2015).

This work is licensed under a Creative Commons Attribution 4.0International License. The images or other third party material in this

article are included in the article’s Creative Commons license, unless indicated otherwisein the credit line; if the material is not included under the Creative Commons license,users will need to obtain permission from the license holder to reproduce the material.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/






http://npg.nature.com/reprintsandpermissions/

http://npg.nature.com/reprintsandpermissions/

http://creativecommons.org/licenses/by/4.0/


an epigenetic regulator emerges as microtubule minus-end ... · received 16 jan 2015 | accepted 23...

Documents